The present invention relates to a method for detecting, in a sample or a culture medium, the presence of bacteria that are resistant to the beta-lactam antibiotics through their ability to produce beta-lactamases.
The beta-lactam antibiotics, which comprise the penicillins, monobactams, cephalosporins and carbapenems, are antibiotics that act by inhibiting the synthesis of the peptidoglycan. These are molecules often used in first-line treatment of serious infections both in general practice and in hospital medicine. The beta-lactamases are enzymes that hydrolyse the beta-lactam ring of the beta-lactam antibiotics. This is the most common mechanism of resistance to beta-lactam antibiotics. According to Ambler (Ambler, R. P. Philos. Trans. R. Soc. London Ser B, 1980, 289, 321-331), the beta-lactamases are classified in 4 groups:
A: penicillinases, including extended broad-spectrum beta-lactamases (ESBLs)
B: metallo-enzymes
C: cephalosporinases
D: oxacillinases
In the enterobacteria, the production of extended-spectrum beta-lactamases (designated ESBLs hereinafter) or of hyperproduced cephalosporinases is currently the principal mechanism of resistance to third-generation cephalosporins (designated C3G hereinafter), such as cefotaxime, ceftriaxone or ceftazidime, which are the oxyimino-cephalosporins. Cefepime, which is the only fourth-generation cephalosporin (designated C4G hereinafter), is hydrolyzed by the ESBLs but not usually by the cephalosporinases. The penicillins are also hydrolyzed, but not the carbapenems. The activity of the ESBLs may be inhibited by clavulanic acid, tazobactam or sulbactam (penicillinase inhibitors). The activity of the cephalosporinases may be inhibited by cloxacillin. The carbapenemases (enzymes that may belong to families A, B and D according to Ambler) are enzymes that hydrolyse the carbapenems but usually also the penicillins and the cephalosporins.
The fast, accurate detection of bacteria that are resistant to the third-generation cephalosporins, producers of ESBLs or of hyperproduced cephalosporinases, or of carbapenemases, is crucial for patient management. It should lead to the establishment of suitable antibiotic treatment. In the absence of production of enzymes that hydrolyse the C3Gs, the latter can be used in first-line treatment, whereas otherwise it becomes necessary to resort to the carbapenems. In this case it is also necessary to determine whether these bacteria produce carbapenemases.
At present, the conventional technique used for detecting beta-lactamase-producing bacteria is based on the implementation of an antibiogram and the application of the double disc synergy test, which makes it possible to demonstrate the restoration of sensitivity to C3Gs in the presence of a penicillinase inhibitor. Despite its efficacy, implementation of this technique requires a preliminary step of culturing and isolating the bacteria, which means a delay of at least 24 hours before the production of beta-lactamases is detectable.
The presence of beta-lactamase-producing bacteria may also be demonstrated by methods of molecular biology, for example using PCR and/or sequencing to detect the genes coding for the beta-lactamases. Although they may allow specific characterization of the genes coding for example for the ESBLs, these laboratory techniques are expensive and take quite a long time because of the preliminary extraction of DNA from complex samples.
There is therefore an urgent need in the field of health care to develop a method that is effective, quick and inexpensive for detecting the presence of beta-lactamase-producing bacteria, in order to detect the presence of the bacterial strains that are resistant to beta-lactam antibiotics.
Nordmann et al. (J. Clin. Microbiol., 2012, 50, 3016-3022) describe a method for detecting ESBL-producing bacteria that involves the use of cefotaxime and a pH colour indicator. This method is based on the fact that hydrolysis of the beta-lactam ring of cefotaxime leads to the formation of a carboxylic acid function and therefore to acidification of the reaction medium. The change in pH of the medium is visualized through the use of a colour indicator.
Despite its efficacy and speed, this method first requires a limiting step of isolating the bacterial strains.
Another colorimetric approach consists of using nitrocefin, a chromogenic cephalosporin, which can be hydrolyzed by all the beta-lactamases, thus causing a colour change from yellow to red (O'Callaghan et al., Antimicrob. Agents Chemother., 1972, 1, 283-288). FIG. 11 illustrates hydrolysis of the beta-lactam ring of nitrocefin by a beta-lactamase. More recently, another chromogenic cephalosporin called HMRZ-86 (Hanaki et al., Antimicrob. Agents Chemother., 2004, 53, 888-889) has been proposed for detecting bacterial strains resistant to C3Gs, based on the same principle.
However, this method once again requires the use of previously isolated bacterial strains to allow colorimetric detection. This method cannot be used directly on complex samples having a coloration that may interfere with colorimetric detection of the hydrolysis of nitrocefin. Once again, this method involves a delay of at least 24 hours between taking a clinical sample and delivering the result.
In recent years, the MALDI-TOF mass spectrometry technique has also been proposed for detecting the products of hydrolysis of the beta-lactam antibiotics after incubation of the latter in the presence of beta-lactamase-producing strains (Hrabák et al. J. Clin. Microbiol. 2011, 49, 3222-3227; Sparbier et al., J. Clin. Microbiol., 2012, 50, 927-937). However, this laboratory technique, which must also be implemented with previously isolated strains, is still expensive and requires qualified personnel for interpreting the mass spectra.
Moreover, Gonçalves et al. (Electrochemistry Communication 38 (2014) 131-133) have an amperometric biosensor for detecting penicillin G by amperometric measurement of the pH change of the medium linked to hydrolysis of penicillin G to penicilloic acid by penicillinase. Since penicillin G and penicilloic acid are not electrically active, amperometric measurement is carried out in the presence of cobalt, which plays the role of redox mediator.
Chen et al. (Int. J. Electrochem. Sci., 7 (2012) 7948-7959) describe a method for detecting cephalexin electrochemically, after its hydrolysis, achieved after an oxygen-free alkaline solution of cephalexin is heated in the dark at 70° C. The products of hydrolysis of cephalexin (cleavage of the C—C═O bond of the β-lactam ring) described in that document were produced under quite specific extreme conditions and cannot be obtained by incubating cephalexin with a beta-lactamase, which in its case cleaves the β-lactam ring at the level of the amide bond.
To date, the prior art does not describe any substrate of the beta-lactamases or any product resulting from hydrolysis by a beta-lactamase having electrically active properties.